發(fā)布時(shí)間：2018-02-28 08:17

  本文關(guān)鍵詞： 弓形蟲 速殖子 緩殖子 應(yīng)激 轉(zhuǎn)化 差異蛋白 細(xì)胞因子　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：剛地弓形蟲(Toxoplasma gondii)是一種專性細(xì)胞內(nèi)寄生的原蟲,可以引起嚴(yán)重的人畜共患寄生蟲病。弓形蟲宿主范圍極其廣泛,可以感染家禽、家畜、伴侶動(dòng)物以及人類在內(nèi)的所有溫血?jiǎng)游�。�?duì)于免疫功能健全的個(gè)體,弓形蟲的感染不表現(xiàn)出明顯的臨床癥狀,蟲體多以半休眠的組織包囊形式在宿主腦部、肌肉及眼部定居,與機(jī)體長期共存。但是包囊并不是無威脅的存在,當(dāng)宿主的免疫力低下時(shí),包囊會(huì)再活化,進(jìn)而由緩殖子轉(zhuǎn)變?yōu)樗僦匙?引起急性感染。因此,弓形蟲速殖子和緩殖子的轉(zhuǎn)化在弓形蟲致病過程中扮演著關(guān)鍵的角色。研究表明,運(yùn)輸應(yīng)激可以降低宿主的免疫力,從而導(dǎo)致多種疾病的復(fù)發(fā),據(jù)此推測(cè),弓形蟲作為一種機(jī)會(huì)性致病病原體,運(yùn)輸應(yīng)激可能誘發(fā)弓形蟲慢性感染小鼠腦內(nèi)包囊的再活化。弓形蟲速殖子和緩殖子轉(zhuǎn)化過程中,多種期特異性蛋白、熱休克蛋白以及代謝酶類的表達(dá)發(fā)生明顯變化,它們對(duì)弓形蟲的生長以及代謝有著重要的調(diào)控作用。因此,本研究針對(duì)弓形蟲速殖子和緩殖子轉(zhuǎn)化過程中可能的差異蛋白進(jìn)行篩選及鑒定。本研究首先建立運(yùn)輸應(yīng)激誘導(dǎo)弓形蟲緩殖子向速殖子轉(zhuǎn)化的動(dòng)物模型,驗(yàn)證了運(yùn)輸應(yīng)激誘使包囊再活化,即由緩殖子轉(zhuǎn)化成速殖子;隨后模擬運(yùn)輸應(yīng)激,利用細(xì)胞因子抗體芯片檢測(cè)小鼠血清中細(xì)胞因子的變化,結(jié)果顯示小鼠外周血血清中γ干擾素(IFN-γ),白細(xì)胞介素(IL-1α),白細(xì)胞介素(IL-4),白細(xì)胞介素(IL-10),白細(xì)胞介素(IL-12)以及巨噬細(xì)胞集落刺激因子(M-CSF)的表達(dá)水平在應(yīng)激后顯著下降;最后利用雙向電泳以及質(zhì)譜鑒定技術(shù)篩選并鑒定出了緩殖子向速殖子轉(zhuǎn)化過程中的47個(gè)鼠源蛋白和15個(gè)弓形蟲蛋白。具體工作包括以下幾個(gè)方面:(1)弓形蟲緩殖子向速殖子轉(zhuǎn)化動(dòng)物模型的建立利用運(yùn)輸應(yīng)激成功誘導(dǎo)弓形蟲腦包囊的再活化,即由緩殖子轉(zhuǎn)變成速殖子。將弓形蟲PRU株慢性感染小鼠模擬運(yùn)輸應(yīng)激3天后,利用Real-time PCR分別檢測(cè)弓形蟲速殖子期特異性基因SAG1和緩殖子期特異性基因BAG1的m RNA相對(duì)表達(dá)水平。結(jié)果顯示在運(yùn)輸應(yīng)激3天后,BAG1的相對(duì)表達(dá)水平下降,SAG1的相對(duì)表達(dá)水平顯著上升,表明運(yùn)輸應(yīng)激誘導(dǎo)包囊的再活化,即從緩殖子向速殖子轉(zhuǎn)化。(2)運(yùn)輸應(yīng)激影響弓形蟲慢性感染小鼠的免疫水平利用弓形蟲緩殖子向速殖子轉(zhuǎn)化的動(dòng)物模型,分別收集運(yùn)輸應(yīng)激前,應(yīng)激1天,應(yīng)激2天,應(yīng)激3天的小鼠外周血血清,利用細(xì)胞因子抗體芯片檢測(cè)小鼠細(xì)胞因子的變化。結(jié)果顯示,IFN-γ,IL-1α,IL-4,IL-10,IL-12以及M-CSF 6種細(xì)胞因子水平隨著應(yīng)激時(shí)間的延長逐漸下降,在應(yīng)激3天后顯著下降。表明運(yùn)輸應(yīng)激降低慢性感染小鼠的免疫水平。(3)弓形蟲緩殖子向速殖子轉(zhuǎn)化差異蛋白的篩選及鑒定利用弓形蟲緩殖子向速殖子轉(zhuǎn)化的動(dòng)物模型,分別收集并純化運(yùn)輸應(yīng)激前,應(yīng)激1天,應(yīng)激2天,應(yīng)激3天的小鼠腦組織包囊,提取包囊總蛋白,利用雙向電泳技術(shù)篩選緩殖子向速殖子轉(zhuǎn)化過程中的差異蛋白并進(jìn)行質(zhì)譜鑒定。結(jié)果顯示,47個(gè)鼠源蛋白和15個(gè)弓形蟲相關(guān)蛋白的表達(dá)量在緩殖子向速殖子轉(zhuǎn)化過程中顯著變化;隨后利用Real-time PCR驗(yàn)證了弓形蟲肌動(dòng)蛋白(Actin),微線體蛋白13(MIC13),致密顆粒蛋白9(GRA9),致密顆粒蛋白1(GRA1)和烯醇酶1(ENO1)的m RNA水平在運(yùn)輸應(yīng)激后顯著上升,與雙向電泳結(jié)果一致,表明緩殖子向速殖子轉(zhuǎn)化過程中多種宿主和蟲體的蛋白表達(dá)發(fā)生變化。本研究利用小鼠運(yùn)輸應(yīng)激模型證實(shí)了應(yīng)激導(dǎo)致機(jī)體免疫力下降;成功篩選并鑒定出了弓形蟲緩殖子向速殖子轉(zhuǎn)化過程中的47個(gè)鼠源蛋白和15個(gè)弓形蟲相關(guān)蛋白,為闡明弓形蟲速殖子和緩殖子轉(zhuǎn)化機(jī)制提供了理論依據(jù)。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic protozoa, can cause serious zoonotic parasitic diseases. Toxoplasma gondii can infect a broad host range, poultry, livestock, all warm blooded animal companion animal and human beings. The immune function of healthy individuals, Toxoplasma infection do not show obvious clinical symptoms, the body in the form of semi dormant tissue cysts in the host brain, eye and muscle settled. But long-term coexistence with the body, cyst is not threatened, when the host's immune system is low, will be activated and the cyst, bradyzoite into tachyzoites that caused by acute infection. Therefore, the transformation of Toxoplasma gondii tachyzoite and bradyzoite of plays a key role in the pathogenesis of toxoplasmosis. The results show that transport stress can reduce host immunity, leading to a variety of diseases We speculated that the recurrence of Toxoplasma gondii as an opportunistic pathogen, transport stress may re activation induced by chronic Toxoplasma infected mouse brain cyst. The transformation process of Toxoplasma tachyzoites and in a variety of stage specific protein, heat shock protein and metabolic enzymes expression changed significantly, with regulation an important role of Toxoplasma growth and metabolism. Therefore, screening and identification of possible differences in protein transformation process of this research and the tachyzoites of Toxoplasma gondii tachyzoites in animal models. In this study we developed transport stress induced bow gondii bradyzoite into tachyzoites, verify the transport stress inducing cyst re activation from tachyzoites into tachyzoites; then simulated transport stress, detecting the changes of cytokines in the serum of mice using cytokine antibody array, results showed that the mice of Zhou Xuexue In the interferon gamma (IFN- gamma), interleukin (IL-1 alpha), interleukin (IL-4), interleukin (IL-10), interleukin (IL-12) and macrophage colony-stimulating factor (M-CSF) expression level decreased significantly after stress; finally by two-dimensional electrophoresis and the screening and identification of mass spectrometry and identified 47 mouse protein tachyzoites into tachyzoites of Toxoplasma gondii and 15 protein. The specific work includes the following aspects: (1) Toxoplasma gondii tachyzoites to establish animal model of tachyzoites reactivation induced cerebral Toxoplasma cysts the use of transport stress, from tachyzoites into tachyzoites. The chronic infection of Toxoplasma gondii PRU strain mice simulated transport stress after 3 days, we detected m RNA of Toxoplasma gondii tachyzoite stage specific gene SAG1 and tachyzoites stage specific gene BAG1 the expression level by using Real-time PCR. The results showed In the transport stress after 3 days, the relative expression level of BAG1 decreased significantly increased the relative expression level of SAG1, showed that the reactivation of transport stress induced cysts, which transformed from tachyzoites to tachyzoites. (2) the transport of animal model of chronic infection of Toxoplasma gondii in mice with immune level arch insect bradyzoite into tachyzoites stress effects were collected before 1 days of transport stress, stress, stress for 2 days, the serum of mice 3 days of stress, the changes of cytokines were detected by cytokine antibody array. The results showed that IFN- gamma, IL-1 alpha, IL-4, IL-10, IL-12 and M-CSF 6 kinds of cytokines decreased gradually with the prolonging of stress time, stress in 3 days decreased significantly. That transport stress reduce chronic infection of mice immune level. (3) insect bow shape using screening and identification of Toxoplasma gondii tachyzoites into proteins to tachyzoite to bradyzoite tachyzoites Animal model of transformation, were collected and purified before 1 days of transport stress, stress, stress and stress for 2 days, 3 days in mice brain cyst, cyst extraction of total protein were identified by 2-DE screening bradyzoite into tachyzoites in the process of protein and differences. The results showed that significant changes in the expression of 47 mouse proteins and 15 proteins related to Toxoplasma tachyzoite bradyzoite in transformation process; then using Real-time PCR to verify the Toxoplasma gondii actin (Actin), microneme protein 13 (MIC13), dense granule protein 9 (GRA9), dense granule protein 1 (GRA1) and enolase 1 (ENO1) m RNA levels were significantly increased in the transport stress, whichisinaccordancewith2 dgel results, showed that the expression of bradyzoite into tachyzoites and host insect bodies during protein change. This study using a mouse model confirmed the transport stress stress machine In the process of transformation of Toxoplasma gondii to tachygonite, 47 mouse proteins and 15 Toxoplasma gondii related proteins were successfully screened out and identified, providing a theoretical basis for clarifying the transformation mechanism of Toxoplasma gondii tachygoni and slowly growing organisms.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7

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本文編號(hào)：1546517